Glucocerebrosidase has greatly reduced activity in patients with Gaucher's disease. In order to better understand the structure-function relationship of this enzyme in both normal and Gaucher tissue, we have undertaken studies of the active-site of the human placental enzyme as the reference to which the mutant species may be compared. Conduritol-Beta-epoxide, a potent specific inhibitor of membrane bound glucocerebrosidase, has been used to probe the active-site of human placental glucocerebrosidase. While inhibition of activity occurred readily, inclusion of 0.1% taurocholate in the incubation buffer decreased the time of inactivation by 50%. Studies to locate the precise amino acid which reacts with the conduritol-Beta-epoxide are in progress using a radioactive form of the inhibitor. In this regard the tryptic peptide map generated during our amino acid sequence studies has been helpful. Inactivation of the enzyme also occurred upon alkylation of a single sulfhydryl group by 4-vinyl pyridine (as judged by amino acid analysis). Inclusion of taurocholate, however, failed to enhance the inactivation. Although alkylation of the sulfhydryl by 4-vinyl pyridine inactivated the enzyme, there was no effect on activity when alkylation was attempted using either iodoacetate or iodoacetamide. One would postulate that the sulfhydryl was near the active-site but not involved in activity. The large bulky pyridine could prevent access of the substrate to the active-site. The identification of the free sulfhydryl is continuing as a part of the amino acid sequence studies of glucocerebrosidase. In addition, we are investigating the effect of phospholipids and sphingolipid-activating protein (SAP-2) on the inactivation of glucocerebrosidase by conduritol-Beta-epoxide and vinyl-pyridine. Do either phospholipids or SAP-2 protect glucocerebrosidase from inactivation? These studies, in addition to defining the active-site, will provide insight into the alteration of catalytic activity of the mutant proteins.